Project Description Epithelial development, integrity, and turnover in the digestive tract depend on signals from adjacent mesenchymal cells, and defective signaling may underlie aspects of disorders such as Inflammatory Bowel Disease, pathologic response to infection, malabsorption, and cancer. Crucial mesenchymal signals include Wnt factors and bone morphogenetic protein (BMP) agonists and antagonists, and recent research implicates various (CD34+, Foxl1+, PDGFRA+, Gli1+) cell types as possible sources. However, the precise identities, overlap, and requirements of these populations remain unclear. Integrated consideration of high- resolution microscopic anatomy, molecular profiling of diverse marker-defined cells, and single-cell RNA analysis has led us to identify 3 distinct mesenchymal cell types in the mouse small intestine. Cells expressing high platelet-derived growth factor receptor A (PDGFRAhi) lie closest to the epithelium and correspond to recently described Foxl1+ telocytes. These cells express abundant BMPs and congregate particularly at the crypt-villus junction, whereas two PDGFRAlo cell populations are anatomically and molecularly distinct: those located near the crypt stem-cell zone that uniquely co-express PDGFRA, tetraspanin CD81, and the BMP antagonist Gremlin1, and cells in the villus lamina propria that lack surface CD81. These data collectively suggest the novel hypothesis that crucial crypt-villus BMP gradients reflect dense sources of agonists at the villus base and antagonists at the crypt base. Specific Aim 1 will test this hypothesis and further resolve the distinctive features and functions of mesenchymal PDGFRAhi and PDGFRAlo cells in the mouse small intestine. Building on preliminary data that unfractionated PDGFRAlo cells support expansion of ISCs in vitro, we will investigate the possibly opposing functions of pericryptal CD81+ and villus CD81- subpopulations. In response to ISC damage, the mesenchyme triggers crypt progenitors to dedifferentiate, likely through the same soluble factors that maintain uninjured ISC. Aim 2 proposes a systematic, unbiased approach to identify the specific cellular sources of key restitutional signals. We will also test the hypothesis that mesenchymal PDGFRAhi and PDGFRAlo cells depend on the intestinal epithelium to provide varying concentrations of the ligand PDGFA for their survival, proliferation, and distinct physiologic functions. ISC and crypt progenitors have counterparts in the stomach epithelium, which turns over more slowly, depends less on Wnt signaling, and does not extend villi. Mesenchymal PDGFRA+ cells are equally abundant in both organs, with likely important similarities and differences in organization and functions. To better understand gastric stem/progenitor cell control and the underlying signaling logic in both organs, Aim 3 will characterize stomach mesenchymal PDGFRA+ cells in the same depth that we have achieved in the intestine. Together, these studies apply innovative experimental approaches to examine specific hypotheses that address fundamental questions in digestive tract biology.